Renal sodium reabsorption in the principal cells of the collecting duct occurs across the epithelial, sodium channel (ENaC) and affects extracellular volume homeostatsis and blood pressure control. In the kidney, two distinct pools of epithelial sodium channels are inserted into the apical membrane of the principal cells of the collecting duct. One pool of channels undergoes maturation and activation by proteases in the trans-Golgi network. The other pool avoids maturation and remains inactive. During maturation, the serine proteases furin and prostasin cleave inhibitory residues from the alpha and gamma subunits of ENaC, activating the channel. The inactive pool may be a target of proteases in pathophysiologic states, like hypertension and nephrotic syndrome. Other serine proteases besides furin and prostasin can activate these channels. Recent evidence suggests plasmin and matrix metalloproteinases activate ENaC. The specific aims of the proposed research are: (1) to identify the key regions and residues within inhibitory peptides derived from the gamma subunit required for ENaC inhibition, and (2) to define the role of plasmin and matrix metalloproteinases in the regulation of ENaC activity. The Xenopus laevis oocyte expression system will be used for these studies. Serial deletions in subunits with mutated furin- and prostasin- sites will be generated so they are not cleaved during maturation. Deletions that result in enhanced ENaC activity in Xenopus oocytes will identify putative inhibitory regions. The effects of synthetic peptide, corresponding to an identified inhibitory region, on ENaC activity will be examined. Synthetic peptides with scrambles and substitutions will determine the importance of specific amino acids, charge, and sequence of the inhibitory region. The effect of plasmin and MMPs on ENaC activity will be examined. Analysis of peptide inhibitors and protease activators of ENaC may provide new potential therapeutic targets for hypertension and nephrotic syndrome. A salt transporter in the kidney helps the body manage fluid balance and prevent fluid retention. Abnormalities in the transporter occur in some forms of high blood pressure and may be involved in a kidney disease that causes leg swelling. Understanding the activators and inhibitors of this channel may help identify new therapies to combat these diseases.